Nutrient strengthening and lead alleviation in Brassica Napus L. by foliar ZnO and TiO2-NPs modulating antioxidant system, improving photosynthetic efficiency and reducing lead uptake

With the anticipated foliar application of nanoparticles (NPs) as a potential strategy to improve crop production and ameliorate heavy metal toxicity, it is crucial to evaluate the role of NPs in improving the nutrient content of plants under Lead (Pb) stress for achieving higher agriculture productivity to ensure food security. Herein, Brassica napus L. grown under Pb contaminated soil (300 mg/kg) was sprayed with different rates (0, 25, 50, and 100 mg/L) of TiO2 and ZnO-NPs. The plants were evaluated for growth attributes, photosynthetic pigments, leaf exchange attributes, oxidant and antioxidant enzyme activities. The results revealed that 100 mg/L NPs foliar application significantly augmented plant growth, photosynthetic pigments, and leaf gas exchange attributes. Furthermore, 100 mg/L TiO2 and ZnO-NPs application showed a maximum increase in SPAD values (79.1%, 68.9%). NPs foliar application (100 mg/L TiO2 and ZnO-NPs) also substantially reduced malondialdehyde (44.3%, 38.3%), hydrogen peroxide (59.9%, 53.1%), electrolyte leakage (74.8%, 68.3%), and increased peroxidase (93.8%, 89.1%), catalase (91.3%, 84.1%), superoxide dismutase (81.8%, 73.5%) and ascorbate peroxidase (78.5%, 73.7%) thereby reducing Pb accumulation. NPs foliar application (100 mg/L) significantly reduced root Pb (45.7%, 42.3%) and shoot Pb (84.1%, 76.7%) concentration in TiO2 and ZnO-NPs respectively, as compared to control. Importantly, macro and micronutrient analysis showed that foliar application 100 mg/L TiO2 and ZnO-NPs increased shoot zinc (58.4%, 78.7%) iron (79.3%, 89.9%), manganese (62.8%, 68.6%), magnesium (72.1%, 93.7%), calcium (58.2%, 69.9%) and potassium (81.5%, 68.6%) when compared to control without NPs. The same trend was observed for root nutrient concentration. In conclusion, we found that the TiO2 and ZnO-NPs have the greatest efficiency at 100 mg/L concentration to alleviate Pb induced toxicity on growth, photosynthesis, and nutrient content of Brassica napus L. NPs foliar application is a promising strategy to ensure sustainable agriculture and food safety under metal contamination.

www.nature.com/scientificreports/precipitate with excess methanol to eliminate impurities.Precipitates were dried on a hot plate at 80 °C temperature for 15 min.The precipitates were annealed at 400 °C for 30 min.

Instrumental analysis
Scanning electron microscope (SEM) (German ZEISS Sigma 300) was performed to analyze nanoparticles surface morphology.The X-ray diffraction (XRD) data for ZnO-NPs and TiO 2 -NPs was obtained using a Bruker D8 Advance X-ray diffractometer, which was equipped with radiation and operated in the 2-theta range of 25-65°.FTIR-Thermo Scientific Nicolet iS20) was done to identify various functional groups in nanoparticles.

Plant materials and growth conditions
A pot experiment was conducted in a natural environment (day/night 23.3/16 °C, relative humidity 39 ± 3%) in a botanical garden at GC University, Faisalabad, Punjab, Pakistan.The pots used for the experiment were cylindrical plastic pots (130 g, 7.8 cm diameter, 8.7 cm height).The experiment was carried out under a completely randomized design (CRD) with three replicates.The soil was spiked with Pb using Lead acetate Pb(C 2 H 3 O 2 ) 2 at a loading rate of 300 mg/kg and incubated for two months for Pb stabilization.Each pot was filled with five kg of spiked soil.Seeds of Brassica napus L. (cv.Super Canola) were purchased from Ayub Agricultural Research Institute, Faisalabad (AARI) and disinfected with hydrogen peroxide (2.5% v/v) solution for 20 min and again washed with distilled water (DW).Seeds were incubated in darkness at 25 °C for three days before sowing in the pot.Seven seeds/pots were sown and after germination thinning was done retaining 3 plants/pots.The recommended dose of NPK (120:80:40 kg/ha) was applied to avoid nutrient deficiency.NPs were applied after one week of germination and before foliar application, NPs were ultra-sonicated for 30 min with distilled water to disperse properly at 0, 25, 50, and 100 mg/L concentrations according to the treatment plan.In total, six sprays were applied to each treatment, and at the same time, control plants were sprayed with DW.During the growing period, the plants were irrigated using tap water with a pH value of 7.42, as measured by a pH meter (Portable Meter Hanna HI-9812-51).

Measurement of growth parameters
After harvesting, growth parameters including height (cm) of the plant (shoot length, root length) and fresh and dry weight were determined after separating the plant into different parts.Plant dry weight was measured after drying the plant parts oven (72 h at 70 °C) and ground into fine powder for further analysis.The length of plant parts was measured with a stainless meter rod and weight with an electric weight balance.

Measurement of Photosynthetic pigment and SPAD values
To determine the concentration of chlorophyll, plant leaves weighing 0.15 g, were crushed.The resulting crushed leaves were then placed into a testing tube (10 mL) containing 4 mL of a buffer solution (1:1 ratio) containing ethanol and acetone.Subsequently, the tube was incubated in darkness for 5 h.The centrifugation was performed at 3000 rpm for 10 min to collect the supernatant and measured by spectrophotometer (Labman LMSPUV1900 Double Beam UV-VIS Spectrophotometer) 33 .Equations used to calculate chlorophyll and carotenoid content: The SPAD value of leaves was determined with a portable SPAD meter (atLEAF CHL STD chlorophyll meter 502).In addition, an Infrared gas analyzer (3051c Plant photosynthesis meter Hangzhou Chincan Trading Co., Ltd) was used to record the leaf gas exchange parameters on a sunny (10: am to 11: am).The calibration of the Portable Infra-Red Gas Analyzer was conducted at different scales, including air temperature (20 °C), relative humidity (60%), and ambient CO 2 concentration (400 ppm).Fully extended second flag leaves were chosen from each pot to measure the stomatal conductance (Gs), transpiration rate (Tr), photosynthetic rate (Pn), and water use efficacy (WUE) was measured.Water use efficiency was calculated as WUE = Pn/Tr.

Determination of antioxidant enzymes and oxidants
To prepare antioxidant enzyme extract, leaf samples were ground using a pre-cooled pestle and mortar in a phosphate buffer solution (PBS) with a pH of 7.8.The homogenized mixture was centrifuged to obtain a supernatant (enzyme extract) for antioxidant enzyme activity analysis.Peroxidase (POD) activity and superoxidase (SOD) activity was measured according to Zhang 34 .For POD activity, enzyme extract was added to PBS and 300 mM of hydrogen peroxide (H 2 O 2 ) and measured with a spectrophotometer.To determine SOD activity the reaction solution was prepared with enzyme extract, L-methionine, riboflavin, and NBT, with EDTA-NA 2 and quantified using a spectrophotometer.Catalase (CAT) activity was measured by Aebi 35 .The APX was determined by following the method of Nakano and Asda 36 .The analysis of malondialdehyde (MDA) in plant tissues was determined according to the method described by Heath and Packer 37 .In brief, the leaf sample (0.5 g) was homogenized with 0.5% thiobarbituric acid (TBA) and added with 20% TCA solution.To determine H 2 O 2 content, the leaf sample was pulverized in a TCA solution by adopting the method proposed by Jana and Choudhuri 38 .The electrolyte leakage (EL) in the leaves was determined according to the modified method described by Dionisio-Sese and Tobita 39 .The EL was calculated by the ratio of the percentage of EC 1 to EC 2 as follows:

Metal and nutrient estimation
To measure Pb concentration, and nutrient content the plant roots and shoot samples (0.5 g) were acid digested with HNO 3 : HClO 4 (3:1 v/v) solution on a hot plate and after digestion filtered through 0.22 µm filters 40 .The filtered samples were subsequently used to measure Pb and nutrient concentrations in the samples using ICP-OES (Optima 7000DV ICP-OES, Perkin Elmer).

Statistical analyses
The data was analyzed using IBM SPSS Statistics (V20) with one-way analysis of variance (ANOVA) on mean values from three replicates and graphs were plotted by MS Excel (version, 2019).Tukey (HSD) test at 95% probability was employed to analyze the significance of the treatments.The alphabets were used to denote significant and non-significance variations among treatments.

ZnO and TiO 2 nanoparticles characterization
SEM analyses showed the size, shape, and structure of the synthesized nanoparticles (NPs).SEM image of ZnO-NPs showed the hexagonal shape, uniform distribution, and nanoscale range at 10.00 k× and 500.00k× magnification as demonstrated by their micrographs, and with particle size 65.29 nm (Fig. 1a-c).The EDX spectra of the NPs demonstrated the presence of Zn and O confirming the purity of obtained NPs as shown in Fig. 1d.ZnO-NPs showed phases in all XRD patterns.All of peaks observed at 2 theta positions were 31.732°,34.364°, 36.207°,47.469°, 56.526°, 62.754°, 67.851°, 68.995° along with their corresponding reflected planes (100), (002), (101), (102), (110), ( 103), (112), (201) respectively with JCPDS No. 01-080-0074.The XRD pattern of prepared ZnO-NPs is shown in Fig. 1e.The FTIR spectra of ZnO-NPs findings are shown in Fig. 1f.The observed peaks indicate the functional group that is typical for the ZnO-NPs.The absorption peaks were determined to lie between 3438.47 cm −1 , 1633.01 cm −1 , 1383.67 cm −1 , 1120.55 cm −1 .The absorption peak at 496.06 cm −1 is due to the presence of metal oxide nature in the Zn-O vibration mode 41 .
Under SEM, TiO 2 -NPs have predominantly spherical-shaped clusters with adequate dispersion, and clean shape and were evenly distributed at 5.00 k× and 50.00 k× magnification with particle size 41.21 nm (Fig. 2a-c).Compositional analysis TiO 2 -NPs of by EDX demonstrated the presence of Ti and O (Fig. 2d).The XRD pattern of TiO 2 -NPs peaks observed at 2 theta positions were 25.335°, 38.611°, 48.104°, 53.921°, 55.138 along with their corresponding reflected planes (101), ( 112), ( 200), ( 105), (211) respectively with JCPDS No. 01-083-2243.The crystal system of the prepared TiO 2 nanostructure is tetragonal given in (Fig. 2e).The FTIR spectrum of titanium oxide nanoparticles are shown (Fig. 2f).The absorption peaks indicate the functional group was determined to lie between the range of 400.63-4000 cm −1 .The broad peak at 3413.59 cm −1 was related to the interaction of the hydroxyl group (-OH) attached to Ti.The 1632.24 peak corresponds to the -OH bending vibration.The broad peak at 400.63 cm −1 is assigned to the bending vibration bonds (Ti-O-Ti) 42 .

Effect of ZnO and TiO 2 nanoparticles on growth parameters
Our results showed that the lowest growth in plants was observed in treatment without any supplementation of NPs (NPs-0) indicating the negative effect of Pb on plant growth.However, TiO 2 -NPs and ZnO-NPs foliar application in dose additive manner enhanced growth rate of Brassica napus L. grown under Pb contaminated soil as shown in (Fig. 3).Specifically, shoot and root length grown in Pb contaminated soil significantly (p ≤ 0.05) enhanced in NPs 100 mg/L treatment by 82.3% and 74.1% in TiO 2 and 75.1% and 63.4% in ZnO-NPs respectively, as compared to control (NPs-0).Similarly, when compared with the control without NPs, the application of TiO 2 -NPs and ZnO-NPs led to significant enhancement in root fresh weight and root dry weight which was 77.7%, 85.3% for 100 mg/L TiO 2 -NPs and 61.4%, 76.6% for 100 mg/L ZnO-NPs treatment respectively, as compared to control (NPs-0).The shoot fresh weight and shoot dry weight dramatically increased up to 68.9% and 89.1% by foliar spray of 100 mg/L TiO 2 -NPs up to 57.8% and 78.7% by application of ZnO-NPs respectively, as compared to control treatment (NPs-0).The maximum number of leaves was also observed in 100 mg/L TiO 2 -NPs 93.3% and 100 mg/L ZnO-NPs 91.5% as compared to control (NPs-0) (Fig. 4).Overall, our results showed that NPs reversed the adverse effects of Pb toxicity and dramatically increased all growth indices compared to plants grown under only Pb stress without NPs treatment.

Effect of TiO 2 and ZnO nanoparticles on photosynthetic pigment
TiO 2 and ZnO-NPs foliar application showed a positive influence on the photosynthetic pigments of plants under Pb stress.The maximum (p ≤ 0.05) increase in photosynthetic activity was observed in 100 mg/L NPs treatment (Fig. 5).An increase in Chlorophyll a was found at TiO 2 and ZnO-NPs 100 mg/L by 66 as compared to control (NPs-0).Likewise, chlorophyll b was increased by 86.3% and 77.3% at higher TiO 2 -NPs and ZnO-NPs (100 mg/L) respectively, as compared to the control.A prominent increase in carotenoid content was also observed at 100 mg/L TiO 2 -NPs followed by 100 mg/L ZnO-NPs with increasing values of 89.7% and 76.8% respectively, as compared to control (NPs-0).The imperative role of TiO 2 and ZnO-NPs was also observed in improving SPAD values.The highest SPAD value was found as 52.09 at 100 mg/L TiO 2 followed by 100 mg/L ZnO-NPs 49.14 and 50 mg/L TiO 2 -NPs (45.4) and 50 mg/L ZnO-NPs (43.9), which was 79.1%, 68.7%, 56.8%, 44.9% higher than control (NPs-0), respectively (Fig. 5).

Effect of TiO 2 and ZnO nanoparticles gas exchange parameters
The results depicted that plants treated with TiO 2 -NPs and ZnO-NPs substantially increased gas exchange parameters.When compared with control (NPs-0) all treatments with NPs boosted gas exchange metrics (

Pb accumulation and fractions in plant parts
The results depicted that Pb accumulation in plant parts diminished with increasing concentration of NPs (Fig. 8).The highest shoot and root Pb concentrations were estimated to be higher at control (NPs-0).In contrast, the lowest Pb concentrations were found where higher concentrations of NPs (TiO

Effect TiO 2 -NPs and ZnO-NPs on nutrient profiling under Pb stress
The lowest micro and macro nutrients were recorded in the root and shoot of Brassica napus L. grown under only Pb stress without NPs application.While, the foliar application TiO 2 -NPs and ZnO-NPs significantly enhanced root and shoot zinc (Zn), iron (Fe), calcium (Ca), manganese (Mn), magnesium (Mg), and potassium (K) concentrations as compared to control (NPs-0) as depicted in (

Foliar application NPs improved plant growth and photosynthetic activity under Pb stress
Lead (Pb) is a non-essential and hazardous metal that hinders the normal growth and productivity of plants consequently threatening food security 43 .Additionally, high levels of Pb can disrupt plants metabolic systems 13,44 .Pb disrupts the enzyme activities that result in alterations in mineral nutrition and membrane permeability, inhibiting plant growth, photosynthesis, and morphological traits 45 .Several studies have highlighted the detrimental effects of Pb toxicity on crop production 12,46,47 .Pb toxicity reduced spinach growth 48 and resulted in retarded germination and low biomass in brassica plants 49,50 .Here in, Pb stress without NPs treatment showed poor growth in terms of shoot and root length, root dry and fresh weight, shoot dry and fresh weight, as well as number of leaves (Figs. 3, 4).The decrease in biomass might be due to the accumulation of Pb which led to excessive ROS generation and damaged plant cell membrane 2 .Pb toxicity inhibits photosynthetic pigment production (Fig. 5) by disrupting the Calvin cycle and carbon fixation in several agricultural plants 51 .Interestingly, TiO 2 and ZnO-NPs application had a positive impact on different aspects of plant health.In particular, the application of these NPs at a 100 mg/L application rate significantly improved plant growth characteristics, including the length of shoots and biomass (Figs. 3, 4), and in coherence with previous studies [52][53][54] .The improvement of biomass could potentially be attributed to the reduction of Pb (Fig. 8) concentration and higher mineral nutrients in NP-treated plants 52 .
The positive impact on growth attributes indicates an overall improvement in the physiological functions of Brassica Napus L. under Pb stress (Fig. 5).Increased Mg, Ca, and K content (Table .2) in NPs treated plants further supported our photosynthetic pigment results.These results are in line with many previous studies 55, 56 .The foliar application of TiO 2 -NPs resulted in a substantial increase in the photosynthetic pigment in wheat and maize crops 53,57 .Earlier research has demonstrated that cowpea plants showed increased chlorophyll concentrations after 100 mg/L foliar application of TiO 2 -NPs 56 .TiO 2 -NPs influence the absorption of minerals such as Mg and Mn.Mg is necessary for the synthesis of chlorophyll, while Fe, Mn, and Zn are vital elements required for pigment production 58 .Due to photocatalytic properties, TiO 2 -NPs can improve photosynthesis efficiency 59 .Our results are also in agreement with a previous study by Chen et al. 3 who reported that 100 mg/L ZnO-NPs foliar application augmented photosynthetic activity by increasing chlorophyll concentration in plants under Cd stress.It has been observed that ZnO-NPs can boost plant Zn concentration with other essential nutrients, and regulate various physiological and biochemical activities 17,52,60 .
Gas exchange parameters act as stress biomarkers of metal stress in plants due to their sensitivity to environmental conditions.Higher Pb concentrations in plant leaves impede photosynthesis, hinder electron transport, and close stomata to reduce CO 2 assimilation 61,62 .The inhibitory effect of Pb on Brassica juncea.L by declining transpiration (Tr) and photosynthetic rate (Pn) has also been observed by Agnihotri & Seth 63 .In our study, it is observed that TiO 2 -NPs and ZnO-NPs foliar application significantly enhanced Brassica napus L. gas exchange parameters (Table 1).It is documented that applying TiO 2 -NPs increased Rubisco activity, a key enzyme in the Calvin cycle that ultimately enhances carbon dioxide fixation and boosts photosynthetic activity in plants 56,64 .Our findings are aligned with previous studies by Ahmad et al. 65 and Alhammad et al. 66 who reported that ZnO-NPs foliar application significantly increased photosynthetic rate, and stomatal conductance under As and Pb stress.

Foliar application of NPs improved plant antioxidant enzyme activity and reduced Pb induced oxidative damage
Numerous studies found that Pb induces oxidative stress in various plant species indicated by higher levels of MDA production 44,67 .MDA is generated during lipid peroxidation and triggered by ROS production 68,69 .Conversely, it is possible that increased levels of antioxidant activity could reduce the oxidative stress induced by HMs by scavenging excessive reactive oxygen species (ROS).High Pb concentration disrupts plant metabolism, lowering antioxidant enzyme production and causing significant harm to the plant defense system, as observed in current results 70 .Our results portrayed that foliar application of TiO 2 and ZnO-NPs reduced oxidative stress by decreasing H 2 O 2 , MDA, and EL content in plants.We observed a substantial increase in POD and CAT activities of plants which plays an important role in reducing oxidative stress and defense mechanism against Pb stress (Fig. 7).SOD scavenges O 2− to H 2 O 2 , while CAT and POD decompose H 2 O 2 into water and oxygen molecules in plant cells 64 .Our results are confirmed by several studies that NPs can reduce the indicators of ROS (MDA,    65 .This decrease in oxidative damage can be attributed to ZnO-NPs interaction with the plasma membrane of cells, leading to the ROS scavenging and protecting the cell membrane against oxidative damage 28 .

NPs ameliorative role in Pb mitigation
Pb primarily enters the root through the root apoplast pathways or calcium ion channels.Studies have demonstrated that there is a higher accumulation of Pb in the roots compared to the shoot depending on the concentration of Pb 12,62 .Our results showed a higher accumulation of Pb in roots followed by shoots in plants grown under only Pb stress (Fig. 8).It is similar to the previous study in Helianthus annuus.L 8 Oryza sativa.L 44 .The observed reduction in Pb concentration in the present study might be related to the augmentation of antioxidant activities and improved biomass in the NPs treated plants, thus resulting in an overall reduction in Pb accumulation in the plants.The current findings for decreased Pb accumulation in plant roots and leaves under NPS exposure are similar to several previous studies 17,50,71 .TiO 2 -NPs exposure to rice plants decreased Pb bioaccumulation by ≥ 80% in a dose-additive manner 72 .The application of TiO 2 -NPs led to a reduction in the levels of Cd in the Mentha piperita L. 71 and wheat plants 73 .Kumar et al. 74 reported that exogenous application of TiO metals and activation of the plant defense system 75 .Similarly, the efficacy of ZnO-NPs in reducing metal toxicity has been documented by previous studies 23,76,77 .Similar to our results previous studies showed that exogenous application of ZnO-NPs effectively reduced Cd accumulation in tomato plants Sun et al. 78 , wheat 23 , and rice 79 by decreasing oxidative damage.ZnO-NPs can enter through stomatal channels to the leaf epidermis and release Zn ions into the apoplast and taken up by mesophyll cells thereby reducing stress induced by HMs in plants 52,75 .

Plant Nutrient Homeostasis by NPs under Pb stress
It is well known that higher Pb significantly hinders the entry of various ions to their respective absorption sites on the roots by competing with other essential nutrients and translocated through the plant system via the xylem vessels in an upward direction, together with other nutrients, and is deposited into the endodermis 80 (Fig. 9).
In present study it is confirmed that plants under Pb stress showed lower concentration of Zn, Fe, Mg, Mn, Ca and K and higher concentration of Pb in root and shoot of Brassica Napus L. indicating that Pb interferes with the movement of nutrients to the above-ground part along with below ground parts as compared to the plants treated with NPs (Fig. 9).However, it has been observed that TiO 2 -NPs and ZnO-NPs significantly transformed the mineral balance (Table 2).A previous study showed that TiO 2 -NPs resulted in increased Zn levels in barley kernels 81 .However, some studies also reported the adverse effects of TiO 2 -NPs with increasing concentration 82,83 .TiO 2 -NPs surface is highly reactive, enhancing root porosity and increasing water and nutrient uptake in both normal and stressful conditions 84 .ZnO-NPs foliar application significantly increases the concentration of nutrients in purslane 77 and foxtail millet 84 .Similarly, in agreement with our results, a recent study by Nafees et al. 18 reported that ZnO-NPs foliar application significantly improved micronutrients and macronutrients in the roots and leaves of spinach under Cd stress.Zinc is a vital nutrient and can improve the plants vascular system leading to strengthened nutritional status 85 .The increase in plant nutrients by ZnO-NPs may also be linked to improved photosynthetic process and reduction of Pb accumulation in Brassica Napus L. which led to higher carbon assimilation and transpiration rate thereby improving nutrient homeostasis and overall plant development.

Figure 3 .
Figure 3.Effect of ZnO-NPs and TiO 2 -NPs on root length (a), shoot length (b), root fresh weight (c), and root dry weight (d) of Brassica napus L. grown under lead contaminated soil.The vertical bar on the graph demonstrates the standard deviation.Data are presented as values of three independent replicates, where each replicate constituted three plants per pot ± SD.Different letters on the bars indicate significant differences among treatments at p ≤ 0.05.

Figure 4 .
Figure 4. Effect of ZnO-NPs and TiO 2 -NPs on leaf fresh weight (a), leaf dry weight (b), and the number of leaves (c) of Brassica napus L. grown under lead contaminated soil.The vertical bar on the graph demonstrates the standard deviation.Data are presented as values of three independent replicates, where each replicate constituted three plants per pot ± SD.Different letters on the bars indicate significant differences among treatments at p ≤ 0.05.

Figure 5 .
Figure 5.Effect of ZnO-NPs and TiO 2 -NPs on chlorophyll a (a), chlorophyll b (b), total chlorophyll (c), carotenoids (d) and SPAD values (e) of Brassica napus L. grown under lead contaminated soil.The vertical bar on the graph demonstrates the standard deviation.Data are presented as mean ± SD (n = 3).Different letters on the bars indicate significant differences among treatments at p ≤ 0.05.

Figure 6 .
Figure 6.Effect of ZnO-NPs and TiO 2 -NPs on catalase (a), peroxidase (b), superoxide dismutase (c), and ascorbate peroxidase activities (d) in leaves of Brassica napus L. grown under lead contaminated soil.The vertical bar on the graph demonstrates the standard deviation.Data are presented as mean ± SD (n = 3).Different letters on the bars indicate significant differences among treatments at p ≤ 0.05.

Figure 7 .
Figure 7. Effect of ZnO-NPs and TiO 2 -NPs on the accumulation of malondialdehyde (a), hydrogen peroxide (b), and electrolyte leakage (c) in leaves of Brassica napus L. grown under lead contaminated soil.The vertical bar on the graph demonstrates the standard deviation.Data are presented as mean ± SD (n = 3).Different letters on the bars indicate significant differences among treatments at p ≤ 0.05.

Figure 8 .
Figure 8.Effect of ZnO-NPs and TiO 2 -NPs on Lead (Pb) content in shoots (a), and Pb in roots (b) of Brassica napus L. grown under lead contaminated soil.The vertical bar on the graph demonstrates the standard deviation.Data are presented as mean ± SD (n = 3).Different letters on the bars indicate significant differences among treatments at p ≤ 0.05.

TiO 2 -
NPs and ZnO-NPs on Brassica napus L. under Pb stress.The foliar application of 100 TiO 2 -NPs and ZnO-NPs significantly enhanced Brassica napus L. growth and physiology.TiO 2 -NPs and ZnO-NPs reduced oxidative stress by upregulation of leaf antioxidant enzyme activity.Furthermore, these NPs significantly reduced Pb uptake in dose dose-dependent manner by improving plant defensive mechanism.Our study demonstrated that the application of NPs (TiO 2 -NPs and ZnO-NPs) enhanced Zn, Fe, Mg, Mn, Ca, and K in Brassica napus L. root and shoot by reducing Pb concentration in plant parts and maintaining ionic balance.Overall study concluded that both TiO 2 -NPs and ZnO-NPs proved to be efficient in mitigating Pb induced toxicity and nutrient strengthening under metal stress.Further studies are needed to focus on gene expression related to Pb uptake in plants by TiO 2 -NPs and ZnO-NPs under different conditions in fields.

Table 1 .
Effect of ZnO-NPs and TiO 2 -NPs on gas exchange parameters of Brassica napus L. grown under lead contaminated soil.Values are the means ± SD (n = 3).Different letters indicate significant differences among treatments at p ≤ 0.05.

Table 2 .
Effect of ZnO-NPs and TiO 2 -NPs on nutritional profile in root and shoot of Brassica napus L. grown under lead contaminated soil.Values are the means ± SD (n = 3).Different letters indicate significant differences among treatments at p ≤ 0.05.